For in-depth product & services help, ask our
Technical Information Scientists
Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chr17:56582929-56583016 88bp TGAGCAAGAGGGCTGTAAAGA AGCAGAGCAGACACCCATTC
Mut= 90 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TAAAGGTTTGCTTTGTTATGGGAGGCCTTTTGAGCGCCCCATTCAGCTCTTTGCCCTGCCTGAATCAAGGACAGGTTTCCTACCATCATCCCTGTGTTTGATGAAAGAGCACACAAGATGAGCGGTCACCAACCCTGATTTGGGGTTAATACTTGGATTTTATTCTATTTTTTTGTCTTGGGTTGGGGATTACATCTAGGGCTACAAGCTGCCAGGTCATGTATGACCACAACCTGctgacagtgactctgcaacccaagctttcagcttccctttctctaggttttgggtcttgagcagggaaggggagctggctgctaaatgtggtcttaagctagattgtgaacacttgcaactgcaaagttgcttcttaaagaacactttgtgtcaacccctcctaaaagataactttatttaaaacttcatactctttcccatcaggcatttaaggaggaaggacaagaacctgaggaggttggaatcagctggggagctgtgagcaagagggctgtaaagagaaacaccaaaggtacttaactgcctgtgcttgacgCTGGGCCTGGGGAATGGGTGTCTGCTCTGCTCTGTGTATGTGTAATATGCATACTTAGATGTGCATGTGTGTGGGACAGCCATAGTGTCACATTGTGTCCACCTTAGGTTTTGTTTTTGTCTTAAGGTTTATTTTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGCGCGCTTGCCTGCATGTGAATGCACATGTGTTTGGTGCCCACAGAGGTCAGAAGCAGGTCTTGGACCCCTT
This mutation is a 317 bp deletion beginning at Chromosome 17 position 56,582,960 bp and ending after 56,583,276 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40784 | TGA GCA AGA GGG CTG TAA AGA | Wild type Forward | A | |||
| 40785 | AGC AGA GCA GAC ACC CAT TC | Common | A | |||
| 40786 | TTG GGT TGG GGA TTA CAT CT | Mutant Forward | A | |||
| 40787 | Fluorophore-1 | CAA AGG TAC TTA ACT GCC TGT GC | Quencher-1 | WT Probe | ||
| 40788 | Fluorophore-2 | ACC ACA ACC TGC TGG GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40784 | 0.40 uM |
| 40785 | 0.40 uM |
| 40786 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.